Development of a high-titer retrovirus producer cell line and strategies for retrovirus-mediated gene transfer into rhesus monkey hematopoietic stem cells.
Retroviral-mediated gene transfer into pluripotent hematopoietic stem cells has been difficult to achieve in large animal models. We have compared several infection protocols in a murine model system and concluded that bone marrow can be maintained and infected in vitro for 2-6 days. We have also developed an amphotropic producer clone that generates greater than 10(10) recombinant retroviral particles (CFU) per milliliter of culture medium. Autologous rhesus monkey bone marrow cells were co-cultured with either high- (2 x 10(10) CFU/ml) or low- (5 x 10(6) CFU/ml) titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone marrow cells from all three animals reconstituted with cells co-cultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow co-cultured with the low-titer producer clone exhibited no evidence of gene transfer.
Bodine
D M
DM
Clinical Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
McDonagh
K T
KT
Seidel
N E
NE
Nienhuis
A W
AW
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D005260:Female; D005814:Genes, Viral; D005821:Genetic Techniques; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D008253:Macaca mulatta; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection
D015345:Oligonucleotide Probes